We have been studying crystallins, the abundant water-soluble proteins responsible for the optical properties of the eye lens, as a model of tissue-specific gene expression. This year we have concentrated on the expression of the small heat shock protein/alphaB-crystallin gene in mice, zebrafish and the blind mole rat. Previous work has shown that tissue-specific alphaB-crystallin gene expression is directed by an enhancer situated approximately in the middle of the 1 kb intergenic region separating the alphaB-crystallin gene and its relative, the MKBP/HspB2 gene. Last year we showed that the glucocorticoid receptor (GR) and Sp1 bind enhancer elements E1 and E3 of the mouse gene, respectively, and differentially affect the divergently arranged promoters. This year we have shown that the aryl hydrocarbon receptor (AhR) and the aryl hydrocarbon nuclear translocator (ARNT) activate the mouse alpha B-crystallin promoter in a dose-dependent, synergistic and ligand-independent manner. While there is an AhR/ARNT binding site in the E2 enhancer element, a truncated promoter lacking the enhancer is still activated by AhR/ARNT, suggesting indirect activation.[unreadable] [unreadable] We have continued investigating expression of the two co-orthologous alphaB-crystallin genes in zebrafish. The alphaB1-crystallin gene is more specialized for lens expression than is the alpha B2-crystallin gene. However, unlike the single mouse alphaB-crystallin gene, there is little if any alphaB1-crystallin gene expression in the lens until later in development (approximately 8 days post fertilization), while the alphaB2 crystallin gene is expressed at low levels in many tissues, including the lens, throughout zebrafish development. Both alphaB1- and alphaB2 crystallin gene expression increases in the lens at 8 days of development and are highly expressed in the adult lens. Promoter activities of the two genes were studied in microinjected zebrafish embryos. AlphaB1 promoter activity was not detected at any developmental stage, while alphaB2 promoter activity was present in skeletal muscle and heart by 3 days of development. [unreadable] [unreadable] We have continued a project on the blind mole rat. This rodent develops an eye during embryogenesis that regresses and has a degenerate lens fragment in the adult. We showed earlier that the truncated (-661/+43) alpha B-crystallin promoter/enhancer fragment of the mouse and blind mole rat (-668/+45) are similar but not identical. Luciferase reporter transgenes driven by either a mole rat or a mouse alphaB-crystallin promoter/enhancer are expressed similarly in the heart of transgenic mice. However, the truncated mole rat promoter/enhancer fragment is at least than 10 times more active than the mouse promoter/enhancer fragment in skeletal muscle and, unlike the mouse promoter fragment, is barely active in the lens of transgenic mice. This year we made two chimeric promoters (mole rat enhancer/mouse proximal promoter and vice versa) and are presently testing their activity in transgenic mice. The chimeric mouse enhancer/mole rat proximal promoter showed low lens expression but high muscle expression (as the wild type mole rat enhancer/promoter). The activity of the chimeric promoter being 2-5 times higher than that of the blind mole rat promoter in every tissue. The chimeric mole rat enhancer/mouse proximal promoter is presently under investigation.[unreadable] [unreadable] Cubomedusan jellyfish havethe most basal sophisticated, lens-containing eyes known. Previously we demonstrated that there are 3 distinct jellyfish lens crystallins (J1-3), that J1 and J3 are expressed in the lens and extracellular tissues, and that the J1 and J3 promoters are activated by the transcription factor PaxB. We have now cloned the J2 cDNA, which encodes a novel protein. The J2-crystallin gene is expressed in lens and nonlens tissues of the jellyfish and its promoter is activated by PaxB. These results support the idea that crystallin recruitment of multifunctional proteins was driven by convergent changes involving Pax (and other) transcription factors in the promoters of non-homologous genes within and between species. Finally, we showed that jellyffish camera-type eyes possess vertebrate-like, ciliated photoreceptors containing rhodopsin related to the vertebrate-type family. cDNA data suggest that jellyfish photoreceptors use a vertebrate-type transduction cascade for vision. Moreover, the jellyfish photoreptors contain a melanin pigment similar to the retinal pigmented epithelial cells of vertbrates. Together, our findings suggest parallel evolution of jellyfish and vertebrate eyes and favor the possibility that the lens-conaining eyes of jellyfish arose by independent recruitment of orthologous genes during evolution.